Method for treating cardiac malfunction

ABSTRACT

A method of treating cardiac malfunction by administering a positive inotropic effect-producing amount of Hypothalamic Inhibitory factor (HIF).

RELATED APPLICATION

This application is a division of application Ser. No. 08/338,264 filedNov. 10, 1994 now U.S. Pat. No. 5,716,937 which is a File WrapperContinuation of U.S. application Ser. No. 07/978,872 filed Nov. 19, 1992now abandoned which is a Continuation-in-Part of U.S. Application Ser.No. 07/450,048 filed Dec. 13, 1989, now abandoned.

BACKGROUND OF THE INVENTION

Digitalis, digoxin, ouabain and related substances are cardiacglycosides derived from plants. The main pharmacodynamic property ofcardiac glycosides is the ability to increase the force of myocardialcontraction in a dose-dependent manner (positive inotropic effect). Themost probable explanation for the direct positive inotropic effect isthe ability of cardiac glycosides to inhibit membrane-bound Na⁺,K⁺-activated adenosine triphosphatase (Na⁺, K⁺-ATPase) (Hoffman, B. F.and J. T. Bigger, Jr., “Digitalis and Allied Cardiac Glycosides” in ThePharmacological Basis of Therapeutics, eds. Goodman and Gilman, p. 732,(1980)). The hydrolysis of adenosine triphosphate (ATP) by this enzymeprovides the energy for the sodium potassium pump.

Relatively little is known about the endogenous regulation of Na⁺,K⁺-ATPase. Catecholamines (Phillis, J. W., Cell, Tissue and OrganCultures in Neurobiology, pp. 93-97 (1978); Horwitz, B. A., Fed. Proc.,38:2170-2176 (1979)), thyroid hormone (Smith, T. J. and I. S. Edelman,Fed. Proc., 38:2150-2153 (1979)), aldosterone (Rossier, B. C., et al.,Science, 12:483-487 (1987)), linoleic and linolenic acids (Bidard, J.N., et al., Biochem. Biophys. Acta., 769:245 (1984); Tamura, M., et al.,J. Biol. Chem., 260:9672 (1985); and vanadium (Cantley, L. C., Jr., etal., J. Biol. Chem., 243:7361-7368 (1978)) have all been linked toeither direct or indirect effects on enzyme activity.

Many researchers have tried to isolate a specific endogenous inhibitorof plasma membrane Na⁺, K⁺-ATPase similar to digitalis or ouabain, butof mammalian origin, by measuring immunoreactivity in plasma, to thedigoxin radioimmunoassay in situations where the inhibitor might beelevated. Klingsmueller et al. found digitalis like immunoreactivity inthe urine of Na⁺-loaded normal human subjects (Klingsmueller, et al.,Klin. Wochenschr., 60: 1249-1253 (1982)). Graves, S. W., et al. made asimilar observation in the plasma of uremic subjects (Graves, S. W., etal., Ann. Intern. Med., 99:604-608 (1983)).

The definitive structure of plasma, urinary or tissue inhibitor of Na⁺,K⁺-ATPase is not known (Haupert, G. T., Jr., in The Na ⁺ K ⁺-Pump, PartB: Cellular Aspects; Skou, J. C., et al., Eds., p. 297-320 (1988)).Furthermore, the degree to which various candidate compounds are truly“digitalis-like” in either structure or function remains controversial,since even those substances characterized in the greatest biochemicaldetail manifest some differences with the cardiac glycosides, digitalisand ouabain (Carilli, C. T., et al., J. Biol. Chem., 260: 1027-1031(1985); Crabos, M., et al., Eur. J. Biochem., 162:129 (1987); Tamura,M., et al., Biochem., 27:4244-4253 (1988)).

For example, the digitalis-like factor (DLF) isolated by Gravescross-reacts with antidigoxin antibodies (Graves, S. W., U.S. Pat. No.4,780,314)). However, DLF has never been shown to be a physiologicinhibitor, as would be expected of an endogenous regulator. Byphysiologic is meant an inhibitor that has a very high binding affinityfor the enzyme; reversibly binds and inhibits; has high specificity forthe membrane Na⁺, K⁺-ATPase; and is responsive to relevant stimuli.

Because of their positive inotropic effect, cardiac glycosides (e.g.,digitalis and ouabain) are unrivaled in value for the treatment of heartfailure. Cardiac glycosides are most frequently used therapeutically toincrease the adequacy of the circulation in patients with congestiveheart failure and to slow the ventricular rate in the presence of atrialfibrillation and flutter.

However, cardiac glycosides have narrow therapeutic indices and theiruse is frequently accompanied by toxic effects that can be severe orlethal. The most important toxic effects, in terms of risk to thepatient, are those that involve the heart (e.g., abnormalities ofcardiac rhythm and disturbances of atrio-ventricular conduction).Gastrointestinal disorders, neurological effects, anorexia, blurredvision, nausea and vomiting are other common cardiac glycoside-inducedreactions.

SUMMARY OF THE INVENTION

This invention relates to Applicant's finding that HypothalamicInhibitory Factor, (HIF), has a positive inotropic effect on cardiacmuscle cells. The invention further relates to Applicant's finding thatHIF is a potent constrictor of pulmonary artery and aortic tissue. Thus,the invention comprises, in one embodiment, a method for producing apositive inotropic effect in a mammalian host by administering to saidhost a positive inotropic effect-producing amount of HIF.

HIF does not manifest the same toxicity profile as the cardiacglycosides. Therefore, therapy of cardiac malfunctions with HIF can beaccomplished with less risk of toxicity to the patient.

This invention further relates to the findings that HIF can beadministered therapeutically to treat cardiac glycoside intoxication,edematous disorders and hypotension. Also, HIF can be used to developspecific therapies to prevent hypertension.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F are graphs produced by a phase contrast microscope videomotion detector showing the effects of ouabain (upper panel) and HIF(lower panel) on cardiac myocyte contractility.

FIG. 2 is a graph plotting the inhibitory effects of HIF (2 units/ml)and ouabain (5 mM) or both on total ⁸⁶Rb⁺ uptake by neonatal rat cardiacmyocytes.

FIG. 3 is a graph plotting the inhibitory effects of increasing doses ofHIF on ouabain-sensitive ⁸⁶Rb⁺ uptake (sodium pump activity) inmyocytes.

FIG. 4 is a fluorescence signal showing the effect of HIF (1 unit/ml) oncytosolic free Ca⁺² content in cultured cardiac myocytes exposed for 30and 60 minutes.

FIGS. 5A-5B are graphs plotting the effects of the alpha blockerphentolamine (PhA, 10⁻⁶M) (top) and nominal zero calcium (bottom) onHIF-induced constrictions of a representative vessel.

FIG. 6 shows the HPLC chromatogram of affinity purified HIF using aWaters Resolve RP C18 column.

FIG. 7A represents tandem mass spectrometry analysis of HIF and ouabain.

FIG. 7B represents tandem mass spectrometry analysis of HIF and ouabain.

FIG. 8A shows the HPLC profile for the naphthoylation reaction yieldingHIF pentanaphthoate (peak A) and HIF hexanaphthoate (peak B).

FIG. 8B shows the HPLC profile for ouabain pentanaphthoate.

FIG. 8C shows the HPLC profile for ouabain pentanaphthoate (peak A) andouabain hexanaphthoate (peak B).

FIGS. 9A-9C compare the HPLC profiles of HIF and ouabainpentanaphthoates using a Vydac C18 HPLC column; FIG. 9A shows HIFpentanaphthoate; FIG. 9B shows ouabain pentanaphthoate; FIG. 9C showscoinjection of HIF and ouabain pentanaphthoates.

FIG. 10A shows the ultraviolet absorbance spectrum for HIFpentanaphthoate.

FIG. 10B shows the circular dichroism spectrum for HIF pentanaphthoate.

FIG. 11A shows the ultraviolet absorbance spectrum for ouabainpentanaphthoate.

FIG. 11B shows the circular dichroism spectrum for ouabainpentanaphthoate.

FIG. 12 shows further HPLC analysis of peak B from FIG. 8A above(representing HIF hexanaphthoate) using a Vydac C18 column.

FIG. 13A shows the ultraviolet absorbance spectrum for peak B1 of theVydac C18 profile of HIF hexanaphthoate.

FIG. 13B shows the circular dichroism spectrum for peak B1 of the VydacC18 profile of HIF hexanaphthoate.

FIG. 14A shows the ultraviolet absorbance spectrum for peak B2 of theVydac C18 profile of HIF hexanaphthoate.

FIG. 14B shows the circular dichroism spectrum for peak B2 of the VydacC18 profile of HIF hexanaphthoate.

FIG. 15A shows the full scan mass spectra of naringinase-hydrolysed HIF.

FIG. 15B shows the full scan mass spectra of intact HIF.

DETAILED DESCRIPTION OF THE INVENTION

A physiologic Na⁺, K⁺-ATPase regulator has been isolated from bovinehypothalamus and has been named hypothalamic inhibitory factor (HIF).(Haupert, G. T., Jr., et al., Am. J. Physiol., 247:F919 (1984). Methodsof isolating HIF are described in detail in Examples I and VII.

Using affinity chromatography and reversed phase HPLC, HIF has beenpurified to homogeneity and its structure characterized. HIF is shownherein to have the same molecular mass as ouabain (584 daltons) and tocontain an L-rhamnose. In addition, chemical ionization studies showthat elimination of the rhamnose produces a daughter ion of the samemass as the aglycone (genin) portion of ouabain. However, when acylderivatives were prepared from authentic ouabain and purified HIF, thetwo compounds were shown to be structurally distinct as measured bytheir HPLC and circular dichroism profiles. These differences areconsistent with the observed differences in pharmacologic effectsbetween the two compounds (Cantiello, H. F., Chen,E., Ray, S. andHaupert, G. T. Jr. Am. J. Physiol. 255: F574-F580 (1988); Hallaq, H. A.and Haupert, G. T. Jr., Proc. Natl. Acad. Sci. USA 86: 10080-10084(1989); Janssens, S. P., Kachoris, C., Parker, W. L., Hales, C. A. andHaupert, G. T. Jr. (in press) J. Cardiovasc. Pharmacol.; Anner, B. M.,Rey, H. G., Moosmayer, M., Meszoely, I., and Haupert, G. T. Jr., Am. J.Physiol. 258: F144-F153 (1990)). HIF satisfies several essentialbiochemical criteria for a physiologic regulator of the Na⁺ pump(Haupert, G. T., Jr. and J. M. Sancho, Proc. Natl. Acad. Sci. USA,76:4658-4660 (1979)). HIF inhibits purified Na⁺, K⁺-ATPase reversiblyand with high affinity (K_(i)=1.4 nM) (Haupert, G. T., Jr., et al., Am.J. Physiol., 247:F919-F924 (1984)). In addition, its effects arespecific (Carilli, C. T., et al., J. Biol. Chem., 260:1027-1031 (1985)).

Purified Hypothalamic Inhibitory Factor (HIF) has now been found to havea positive inotropic effect on cardiac muscle cells (i.e., myocytes), asmentioned above. “Positive inotropic effect” means that thecontractility of the cells is enhanced in a dose-dependent manner.

A positive inotropic effect-producing amount of HIF can be administeredto a “mammalian host” (e.g., a human) to treat cardiac malfunction(e.g., congestive heart failure, paroxysmal atrial tachycardia, atrialfibrillation and flutter). Administration can be either enteral (i.e.,oral) or parenteral (e.g., via intravenous, subcutaneous orintramuscular injection). In addition, as HIF does not manifest the sametoxicity profile as the cardiac glycosides, therapy of cardiacmalfunctions with HIF can be accomplished with less risk of toxicity tothe patient.

FIGS. 1A-1F are graphs showing the positive inotropic effects onmyocytes exposed to 1 unit/ml of HIF in comparison to 5×10⁻⁷M ouabain;and in comparison to the toxic effects on myocytes exposed to 1×10⁻⁶Mouabain. In this experiment, one unit of HIF is defined as the amountnecessary to inhibit 1 μg of highly purified Na⁺, K⁺-ATPase by 50% understandard assay conditions at 37° C. (Haupert, G. T., et al., Am. J.Physiol., 247:F919-F924 (1984)). See Example VI.B. for a more detaileddiscussion of the definition of HIF units of activity. The protocol forthe contractility experiments is set forth in Example III C.

In general, the amplitude of contraction (i.e., degree of positiveinotropy) was measured in single, beating myocytes as the amplitude ofsystolic motion (ASM) using a phase contrast video motion detectorsystem. The same detector system was used to measure toxicity, which isevidenced as a decrease in ASM, a change in the position of maximalrelaxation (MR), and an increase in beating frequency.

FIG. 1B shows that 5×10⁻⁷M ouabain, a maximally inotropic but non-toxicdose in these cells, increases amplitude of systolic motion (ASM) by 41%and caused a decrease in beating frequency compared to the same cells inthe control period (FIG. 1A). There was no change in the position ofmaximal relaxation (MR), indicating “therapeutic range” but non-toxiceffects.

FIG. 1C shows that a 1 μM concentration of ouabain causes a decrease inASM, an elevation in the position of MR, and an increase in beatingfrequency, all indicating “toxic range” effects of this only 2-foldhigher dose on the contracting myocytes, reflecting the narrowtherapeutic index of cardiac glycosides.

FIG. 1E shows that 1 unit/ml HIF causes a 37±3% increase in ASM comparedto control (FIG. 1D), with a decrease in beating frequency and no changein the position of MR. Therefore, 1 unit/ml is within the therapeuticrange, but exhibits no toxic effects.

FIG. 1F shows the results following a 1 minute “washout”, during whichthe same cell was perfused with HIF-free buffer. ASM and beatingfrequency returned to control levels indicating rapid reversibility ofthe positive inotropic effects caused by HIF.

HIF effects are more readily reversible than those of ouabain, since a 5minute washout period in ouabain-treated neonatal rat myocytes isrequired to return enhanced contractility to control levels (Werdan, K.,et al., Biochem. Pharmacol., 33:1873-1886 (1984)).

In addition to its use in treating cardiac malfunction, a pharmaceuticalcomposition of HIF can be administered (e.g., enterally or parenterally)to treat patients with serious or life-threatening cardiac glycosideintoxication. Currently, cardiac glycoside intoxication is treatedeither generally by administering potassium or antiarrhythmic drugs tothe patient, or specifically by administering antibody fragments tospecific cardiac glycoside preparations. Patients with severe toxicitymay be unresponsive to general methods of treatment. In addition,although treatment with antibody fragments does neutralize cardiacglycosides in circulation, the antibodies may not effect cardiacglycosides that are bound to cardiac tissue. Furthermore, becauseantibodies are proteins, they are administered intravenously and cancause allergic reactions.

In contrast, HIF not only blocks circulating cardiac glycosides frombinding to the Na⁺, K⁺-ATPase, but also elutes or “chases” previouslybound cardiac glycoside from Na⁺, K⁺-ATPase, presumably by competingwith or interfering with the cardiac glycoside binding site. “Chase”experiments were performed using an assay system whereby purified Na⁺,K⁺-ATPase is reconstituted into liposomes (Anner, B. M. and M.Moosmayer, Biochem. Biophys. Res. Commun., 129:102-108 (1985)). Thedetailed protocol for these experiments is set forth in Example IV, andresults presented in Table 3. In general, liposomes containingfunctional Na⁺, K⁺-ATPase molecules were incubated with ³H-ouabain whichpermits measurement of specific ouabain binding to its binding site onthe Na⁺, K⁺-ATPase. The liposome-Na⁺, K⁺-ATPase-ouabain complex was thenexposed to varying doses of HIF for 10 minutes at 25° C. The bound³H-ouabain was eluted from the Na⁺, K⁺-ATPase by HIF in a dose-dependentmanner, with complete elution of the bound ouabain at an HIFconcentration of 0.5 units per 2.5 microliters liposomes.

Thus, HIF is not only able to prevent digitalis compounds from bindingto the Na⁺, K⁺-ATPase, but, as shown by these experiments, is able todisplace cardiac glycoside already bound to the Na⁺, K⁺-ATPase.Therefore, treatment of cardiac glycoside intoxication with HIF couldserve as a highly specific therapy to rapidly reverse the toxic effectson the heart. In addition, as a non-peptide, oral administration of HIFis possible.

HIF can also be administered (e.g., enterally or parenterally) to treatblood pressure abnormalities. Studies have shown that excess ofendogenous circulating inhibitor of Na⁺, K⁺-ATPase may be responsiblefor essential hypertension in some or many patients. (DeWardener, H. E.and G. W. MacGregor, Kidney Int., 18:1-9 (1980)). Presumably, theincreased intracellular calcium ion concentration resulting from thebinding of an inhibitor to Na⁺, K⁺-ATPase produces blood vesselconstriction and hypertension (Blaustein, M. P., Am. J. Physiol.,232:C165-C173 (1977)).

Experiments were conducted to determine the vasoconstrictive propertiesof HIF. The protocol for these experiments is described in greaterdetail in Example V. In general, Sprague-Dawley rats were anesthetizedand the abdominal aorta surgically removed. 2 mm vascular rings wereattached to a force transducer and bathed in buffer, and tensionadjusted to 1.5 g. Tissue viability was documented and vasoconstrictiveresponses calibrated using known vasoconstrictors such as potassiumchloride and norepinephrine. Blood vessels thus prepared were thentested with varying doses of HIF.

HIF produced potent, reversible vasoconstriction of the vessels, andthese responses were dose dependent. Vessels remained completely viableafter exposure to HIF, documenting absence of toxic effects. Maximumvasoconstrictive responses were similar to those produced by the knownvasoconstrictor substances used as standards. Hypotension, abnormallylow blood pressure, can be caused by low cardiac output, inadequatevascular constriction, or both occurring simultaneously. Since HIF hasbeen demonstrated to both increase the strength of cardiac cellcontraction and promote blood vessel constriction, its administration intherapeutic amounts would be an effective treatment for hypotension.

Experiments were further conducted to determine the vasoconstrictiveeffects of HIF on Sprague-Dawley rat and spontaneously hypertension rat(SHR) pulmonary artery tissue and abdominal aorta tissue. The protocolfor these experiments is described in greater detail in Example VI. Ingeneral, Sprague-Dawley rats or SHR were anesthetized and the pulmonaryartery (PA) and abdominal aorta (AO) surgically removed. 2-3 mm vascularrings were cut from these tissues, attached to a force transducer andbathed in buffer. The tension in the transducer was adjusted to 1.5 g.Tissue viability was documented and vasoconstrictive responsescalibrated using known vasoconstrictors such as potassium chloride andnorepinephrine. The response to 20U (˜4 nM) HIF of blood vessels thusprepared were then compared.

HIF constricted PA rings of hypertensive rats to a significantly greaterextent than PA rings of normotensive rats. However, this difference wasnot observed in abdominal aortic rings. In addition, no significantdifference in HIF-induced contractions was observed between aortic andpulmonary artery rings in normotensive animals, but in hypertensiveanimals the effect of HIF was significantly greater in the pulmonaryartery rings compared to the aortic rings. In all cases, contraction wasabolished by phentolamine, but was unaffected by calcium channelblockade using verpamil. In addition, HIF-induced tension developmentrequired external Ca²⁺.

HIF can also be used to develop specific therapies to prevent excessivevasoconstriction and resulting hypertension. Such therapies wouldinclude but not be limited to: (1) Administering antibodies to HIF forpassive immunizations; (2) administering immunogenic forms of HIF foractive immunity against hypertension; and (3) administering analogues ofHIF which could prevent or modulate binding of native HIF to and actionon the vascular cell Na⁺, K⁺-ATPase.

In addition, by potently inhibiting the Na⁺, K⁺-ATPase activity of renaltubular cells and thereby promoting sodium excretion, a pharmaceuticalcomposition of HIF can be used as a natural diuretic, to promoteexcretion of excess salt and water by the kidneys in patients sufferingfrom such common clinical conditions as congestive heart failure,cirrhosis of the liver, and nephrotic syndrome. Because of the specificinhibitory effect that HIF has on Na⁺, K⁺-ATPase, diuretic therapy withHIF can be accomplished without the side effects (e.g., impotence,rashes, blood lipid abnormalities) which commonly occur with existingdiuretic drugs.

This invention is illustrated further by the following examples, whichare not to be construed as limiting in any way.

EXEMPLIFICATION I. Preparation of HIF

The Na⁺, K⁺-ATPase inhibitor was prepared from bovine hypothalamus aspreviously described (Carilli, C. T., et al., J. Biol. Chem.,260:1027-1031 (1985)). Hypothalami collected fresh and frozenimmediately on dry ice were thawed, homogenized and extracted inmethanol:water (4:1, v:v). Methanol was removed by flash evaporation,and lipids removed by extraction of the remaining aqueous phase withpetroleum ether and chloroform. Initial separation of HIF was carriedout using lipophilic gel chromatography (Carilli, C. T., et al., J.Biol. Chem., 260:1027-1031 (1985)). Further purification wasaccomplished using successive cation and anion exchangechromatographies. Approximately 100 units HIF from the lipophilic gelchromatographies was dissolved in 10 ml doubly distilled water (ddH₂O),applied to a small column containing 10 ml of sulfonic acid cationexchange resin in the protonated form (Amersham, IR 120), and elutedwith ddH₂O at 1 ml/minute. The effluent was collected, lyophilized, theresidue taken up in 1 ml ddH₂O and applied to a column containing 1.5 mlAmberlite IR 958 (Schweizerhall, Inc.) in the bicarbonate form. Thecolumn was eluted successively with two bed volumes ddH₂O. An aliquot ofthe eluate was assayed for specific Na⁺, K⁺-ATPase inhibitory activity(HIF) in a coupled-enzyme assay (Haupert, G. T., et al., Am. J.Physiol., 247:F919-F924 (1984)) and a human erythrocyte ⁸⁶Rb⁺ uptakeassay (Carilli, C. T., et al., J. Biol. Chem., 260:1027-1031 (1985)) todetermine the concentration of HIF activity. The cation exchange stepsuccessfully removed small amounts of gamma amino benzoic acid, serine,threonine and glutamic acid, and the anion exchange step, trace amountsof lactate, all of which had been detected by mass spectroscopicanalysis of active fractions following the lipophilic gelchromatographies. Recovery of HIF was quantitative following theion-exchange steps. HIF following the above procedures is free ofvanadate (emission spectroscopy), NH₄ ion (reductive amination, Sigmakit 170-A), and free fatty acids and lysophospholipids (gaschromatography-mass spectroscopy) which have been shown to interfere inpertinent bioassays.

II. Preparation of Cardiac Cells

Nyocardial cells were isolated from ventrical fragments of the hearts of1-day old Sprague-Dawley rats by serial trypsinizations in a Ca²⁺ andMg²⁺-free Hanks Buffered Salt Solution (HBSS) as previously described(Yagev, S., et al. In Vitro, 20:893-898 (1984)). Trypsinized cells weredecanted into HamF10 medium containing 20% serum and antibiotics andcentrifuged at 1000 r.p.m. for 10 minutes. The cell pellet wasresuspended in HamF10 medium containing fetal calf serum and 10% horseserum with 0.1% penicillin-streptomycin, and diluted to a concentrationof 5×10⁵ cells/ml. For measurements of cytosolic free calciumconcentration ([Ca²⁺]i), the cells were plated on rectangular glasscoverslips (13×30 mm) and for measurements of cell contractility, platedon circular glass coverslips (12 mm), and both types of coverslipsplaced inside petri dishes. For measurements of ⁸⁶Rb⁺ uptake cells wereplated in petri dishes (1−1.5×10⁶ cells/35 mm dish). All cultures wereincubated in humidified 5% CO₂, 95% air at 37° C. Confluent monolayersin which an estimated 80% of cells exhibited spontaneous synchronouscontractions developed by three days, at which time experiments wereperformed.

III. Physiologic Effects of HIF on Cardiac Cells

A. ⁸⁶Rb⁺ Influx Measurements

Na⁺ pump activity was estimated in the cultured cardiac cells as thedifference in ⁸⁶Rb⁺ uptake observed in the absence and presence of 5 mMouabain, following the method of Panet et al. (Panet, R., et al., J.Memb. Biol., 70:165-169 (1982)). To insure saturation binding, myocyteswere preincubated for 20 minutes with HIF (2 unit/ml) or ouabain (5 mM)or both prior to addition of ⁸⁶Rb⁺ to run the 10 minute flux. Myocytemonolayers were washed with HEPES buffer solution (final concentrations,mM: NaCl 150, RbCl 5, HEPES-Tris 10 (pH7.0), CaCl₂ 1, MgCl₂ 5, glucose10), and the uptake initiated by covering over with 0.5 ml of samesolution pre-warmed to 37° C. and containing 2 μCi ⁸⁶RbCl. Incubationswere continued for up to 10 minutes (period of linear uptake of theisotope) (Heller, M., et al., Biochem. Biophys. Acta, 939:595-602(1988)), and the uptake terminated by aspiration of the reaction mixturefollowed by two rapid rinses with 3 ml ice-cold MgCl₂ (125 mM) and twowith 5 ml ice-cold NaCl (165 mM). The cells were lysed with 0.6 ml of0.1 N NaOH containing 0.1% (w/v) sodium dodecylsulfate, and theradioactivity counted in Instagel (Packard) scintillation medium.

FIG. 2 illustrates the inhibitory effects of HIF (2 units/ml) andouabain (5 mM) or both on total ⁸⁶Rb⁺ uptake by neonatal rat cardiacmyocytes. Na⁺ pump mediated uptake (600 nmol/mg protein/10 minutes) iscalculated as the difference in total uptake, and uptake in the presenceof 5 mM ouabain.

Ouabain (5 mM) decreased uptake from control levels of 810 nmol/mgprotein to 210 nmol/mg (74%), while HIF (2 units/ml) decreased theuptake from control levels to 377 nmol/mg (54%). The combination ofouabain plus HIF was not additive, indicating that HIF inhibitoryeffects are specific for K⁺ transport through the Na⁺, K⁺-ATPase, as hasbeen previously shown for human erythrocytes (Carilli, C. T., et ., J.Biol. Chem., 260:1027-1031 (1985)) and renal tubular cells (Cantiello,H. F., et al., Am. J. Physiol., 255:F574-F580 (1988)). HIF (2 unit/ml)thus inhibited ouabain-sensitive K⁺ transport in myocytes by 74%.

The concentration dependence of HIF inhibition was determined bypreincubating cells with HIF at 0.5 units/ml, 0.8 units/ml, 1 unit/ml, 2units/ml and 4 units/ml concentration for 20 minutes. ⁸⁶RbCl (2μCi/well) was then added to run the flux.

FIG. 3 shows the concentration dependence of HIF inhibition ofouabain-sensitive RB⁺ uptake (a specific measure of Na⁺, K⁺-ATPaseactivity) in neonatal rat cardiac myocytes. Values are mean±SEM for n=4determinations at each concentration. HIF-mediated Na⁺ pump inhibitionis dose-dependent and related to the amount of time that the myocytesare exposed to the inhibitor. Ninety percent of Na⁺ pump activity in theneonatal rat myocytes is inhibited by the maximal dose of HIF (4units/ml). FIG. 3 (inset) illustrates the inhibition of active K⁺transport as a function of the time that myocytes are exposed to HIF (2units/ml). Maximal inhibitory effects (i.e., inhibition of myocyte Na⁺,K⁺-ATPase activity by about 90%) required 20-30 minutes preincubationwith HIF.

However, significant pump inhibition also occurred after shorterexposures to HIF. The ID₅₀ for pump inhibition in neonatal rat myocytesoccurs at an HIF concentration of about 0.5 units/ml. This isapproximately 30 fold less than that for cultured porcine renal tubularcells (Cantiello, H. F., et al., Am. J. Physiol., 255:F574-F580 (1988)),therefore suggesting that neonatal rat myocytes have a relatively higheraffinity for HIF.

B. Measurement of Cytosolic Free Ca²⁺ ([Ca²⁺])

Changes in [Ca²⁺]i were detected using the fluorescent probe, fura-2(Grynkiewicz, G., et al., J. Biol. Chem., 260:3440-3450 (1985)).Rectangular glass cover-slips with attached myocytes were placed inbuffered salt solution (BSS, containing in mM: NaCl 140, KCl 5, CaCl₂ 1,MgCl₂ 1, glucose 10, Na₂HPO₄ 1, Hepes-Tris (pH 7.4) to which was added 5μM fura-2/AM, and incubated for 1 hour in humidified 5% CO₂−95% air at37° C. Additional loading medium was added and incubation continued for15 minutes to complete the hydrolysis of fura-2/AM. The cells werewashed and incubated an additional 30 minutes in BSS. Coverslips withloaded myocytes were inserted into a thermostrated (37° C.) cuvettecontaining 2 ml BSS and various additions of HIF or ouabain asindicated. The fluorescence was continuously recorded using a PTIDeltaScan 1 spectrofluorometer. Dual excitation wavelengths alternatedrapidly (60 Hz) between 340 nm and 380 nm, emission wavelength 505 nm.Values of [Ca²⁺]i were calculated from the ratio R=F₃₄₀/F₃₈₀ using theformula: [Ca²⁺]i=Kd B (R−R_(min))/(R_(max)−R), where K_(d) is 225 nM.R_(max) and R_(min) were determined in separate experiments usingdigitonin to equilibrate [Ca²⁺]i with ambient [Ca²⁺] (R_(max)) andaddition of MnCl₂ (0.1 mM) and EGTA (1 mM) (R_(min)). Backgroundauto-fluorescence was measured in unloaded cells and subtracted from allmeasurements.

FIG. 4 shows the effect of HIF (1 unit/ml) on cytosolic free calcium ion([Ca²⁺]i) content in cultured cardiac myocytes. Fluorescence of fura-2loaded myocytes incubated in Buffered Salt Solution (BSS) was recordedcontinuously. Control value (138 nM) is for cells in BSS prior toaddition of HIF (arrow). Recordings are shown after 30 minutes and 60minutes exposure to HIF. Cytosolic free Ca²⁺ concentrations increasedfrom a baseline of 138 nM to 250 nM and 432 nM after 30 and 60 minutesexposure to HIF, respectively.

The onset of change in [Ca²⁺]i caused by HIF occurred within 15 minutes,reached a new steady state concentration by 60 minutes, and remainedstable at this level for at least 2 hours.

The biochemical events underlying cardiac glycoside toxicity and thenarrow therapeutic index characteristic of these drugs is incompletelyunderstood, although excessive intracellular free Ca²⁺ secondary topersistently elevated intracellular Na⁺ associated with tonic pumpinhibition has been postulated as having a central role (Tsien, R. W.and B. U. Carpenter, Fed.Proc. Fed. Am. Soc. Exp. Biol., 37:2127-2131(1978)).

Table 1 shows increases in steady-state [Ca²⁺]i induced by various dosesof HIF. A dose-response relationship was found with 0.5 unit/ml HIFincreasing [Ca²⁺]i in the myocytes to a level (303±15 nM) similar tothat caused by 1 μM ouabain (287±15 nM) under the same experimentalconditions. HIF (1 unit/ml) raised [Ca²⁺]i to a level significantlygreater than that induced by 1 μM ouabain.

TABLE 1 Changes in steady-state cytosolic free Ca²⁺ concentrations([Ca²⁺]i) in cardiac myocytes treated with ouabain or variousconcentrations of HIF* Ouabain HIF (units/ml) Condition Control 10⁻⁶ M0.25 0.5 1.0 [Ca²⁺]i(nM) 138 ± 3 287 ± 15 197 ± 9 303 ± 15 432 ± 18*Values are mean ± SEM; n = 3 determinations at each concentration

While 1 μM ouabain raises the intracellular concentration of Ca²⁺ to287±15 nM, and causes clear toxicity (FIG. 1C), 1 unit/ml of HIF raisesthe intracellular concentration of Ca²⁺ in the same cells to a muchhigher level, 432±18 nM, but is accompanied by a stable, maximalinotropic effect with no sign of toxicity (FIG. 1E).

C. Measurements of Myocyte Contractility

Contractility, determined as amplitude of systolic cell motion (ASM),and beating frequency were measured in individual cells using a phasecontrast microscope video motion detector system according to the methodof Barry, et al., (Barry, W. H., et al., Circ. Res., 56:231-241 (1985)).A glass coverslip with attached cultured myocytes was placed in achamber provided with inlet and exit ports for medium perfusion. Thechamber was enclosed in a Lucite box at 37° C. and placed on the stageof an inverted phase contrast microscope. The cells were covered with 1ml medium containing HIF or ouabain. During continuous perfusion, mediumbathing a cell in the center of a coverslip exchanged with a timeconstant of 15 seconds at a flow rate of 0.96 ml/minute. Image wasmagnified using a 40× objective, and motion monitored by alow-light-level TV camera attached to the microscope and calibrated with262 raster lines. The motion detector monitors a selected raster linesegment and provides new position data every 16 msec for an image borderof a microsphere within the cell layer moving along the raster line. Theanalog voltage output from the motion detector is filtered at low passfilter and calibrated to indicate actual μm of motion, and thederivative is obtained electronically and recorded as velocity of motionin μm/sec. Rate, amplitude and velocity of contraction remained stablefor several hours during control perfusions. The changes incontractility induced by ouabain or HIF were calculated in comparisonwith the contractility of the same cells before addition of ouabain orHIF.

Table 2 summarizes changes in ASM and beating frequency as a function ofvarious doses of HIF. Increasing concentrations of HIF causedprogressive increase in ASM and decrease in beating rate. Maximalincrease in ASM occurred at an HIF concentration of about 0.5 units/ml(39±6%), a level equal to the maximal, non-toxic dose of ouabain (5×10⁻⁷M)

TABLE 2 Effects on amplitude of systolic motion (ASM) and beating ratein cardiac myocytes by various concentrations of HIF, and ouabain (Ou)*HIF (units/ml) Ou (M) 0.2 0.25 0.33 0.5 1.0 5 × 10⁻⁷ ASM, 15 ± 2 22 ± 132 ± 9 39 ± 6 37 ± 3 41 ± 3 % increase Rate, 12 ± 1 15 ± 1 25 ± 7 40 ± 249 ± 6 23 ± 2 % decrease *Values are mean ± SEM; n = 3 for eachconcentration

IV. HIF Ability to Displace Cardiac Glycosides from Their Binding Siteon the Na⁺, K⁺-ATPase

“Chase” experiments were performed using an assay system wherebypurified Na⁺, K⁺-ATPase is reconstituted into phosphatidylcholineliposomes. ATP-filled liposomes containing dispersed, randomly orientedNa⁺, K⁺-ATPase molecules were prepared by the cholate-dialysis methodaccording to Anner (Anner, B. M. and M. Moosmayer, Biophms. Res. Commun.129:102-108 (1985)). Using this miniaturized, two-sided test systemApplicant has also shown that Na⁺, K⁺-ATPase inhibition by a single doseof 0.1 U HIF (approximately 75 fmol), and the membrane permeation of 1.0U HIF (approximately 750 fmol) became measurable, and that an estimationof the minimal number of HIP molecules per unit could be made (Anner, B.M., Rey, H. G., Moosmayer, M., Meszoely, I. and Haupert, G. T. Jr., Am.J. Physiol. 228:F144-F153 (1990)). Despite testing of numeroussubstances including other proposed endogenous Na⁺, K⁺-ATPaseinhibitors, HIF is the only compound (besides the cardiac glycosidesthemselves) tested thus far in the purified system that displays suchstriking transport inhibition.

For “chase” experiments liposomes containing functional Na⁺, K⁺-ATPasemolecules were incubated for 10 minutes at 25° C. with ³H-ouabain (10μM), following which HIF was added in various doses and the incubationcontinued for 10 minutes at 25° C. Reactions were quenched by additionof 125 μl ice-cold stop solution, and the samples applied and elutedthrough (0° C.) a 20-cm Sephadex G-50 medium column which permitsseparation of bound from free ³H-ouabain. Bound ³H-ouabain was elutedfrom the Na⁺, K⁺-ATPase by HIF in a dose-dependent manner as shown inTable 3.

TABLE 3 Elution of ³H-ouabain bound to 2.5 μl Na⁺, K⁺-ATpase liposomesby various doses of HIF. +HIF (units/ 2.5 ul liposomes) −HIF 0.125 0.250.5 ³H-ouabain bound (cpm) 2902 178 111 45 ³H-ouabain eluted (%) — 94.096.2 98.5

V. Vasoconstrictive Properties of the Hypothalamic Na⁺, K⁺-ATPaseInhibitor: The Effects of HIF on Vasoconstriction of Sprague-Dawley RatAbdominal Aorta Rings

Sprague-Dawley rats weighing 250-350 grams were anesthetized withpentobarbital intraperitoneally, the abdominal aorta rapidly excised anddissected free of all loose connective tissue in cold Krebs-Henseleitbicarbonate buffer of the following composition, mM: NaCl, 118.3; KCl,4.7; MgSO₄, 1.2; KH₂PO₄, 1.2; CaCl₂, 2.5; NaHCO₃, 25.0; Na-EDTA, 0.016;and glucose, 11.1. 2-3 mm (length) vascular rings were excised, attachedto a force transducer and bathed in a water jacketed (37° C.) organchamber containing 5 ml of the above buffer gassed with 95% O₂ and 5%CO₂. Isotonic force measurements were obtained with a Grass forcedisplacement transducer attached to a Grass 79D polygraph DC amplifier,and recorded with a two-channel Kipp Zonen recorder. Calibration studiesrevealed that aortic rings placed under 1.5 g tension generated amaximal contractile response after KCl depolarization, and all ringswere therefore equilibrated under 1.5 g tension prior to startingexperiments. Tissue viability for individual experiments was documentedusing known vasoconstrictors such as potassium chloride andnorepinephrine. Blood vessels thus prepared were then tested withvarying doses of HIF, and the magnitude of response compared withKCl-induced contractions. HIF produced potent, reversiblevasoconstrictions of the vessels, and these responses were dosedependent as shown in Table 4.

TABLE 4 Effects on vascular tension in rat abdominal aortic rings byvarious concentrations of HIF, and KCl, shown as increase above restingtension of 1.5 g Change in Tension in Rat Aorta HIF (units/ml) KCl (mM)0.1 0.1 0.4 0.8 20 % increase 2 5 12 26 20

Vessels remained completely viable after exposure to HIF as judged bypreservation of KCl responses following washout of HIF; documentingabsence of toxic effects. Maximum vasoconstrictive responses weresimilar to those produced by the membrane depolarizing dose of KCl.These studies confirm that HIF is a potent vasoconstrictive substance,compatible with its proposed role in regulation of vascular tone andpotential role in the pathogenesis of hypertensive disease.

VI. Vasoconstrictive Properties of the Hypothalamic Na⁺, K⁺-ATPaseInhibitor: The Effects of HIF on Vasoconstriction of Sprague-Dawley Ratand Spontaneously Hypertensive Rat Pulmonary Artery Rings and AbdominalAorta Rings

Spontaneously hypertensive rats show a moderate though significantdegree of pulmonary hypertension which is not secondary to the systemichypertension (Janssens, S. P., Thompson, T. B., Spence, C. R., Hales, C.A., Am Rev Resp Dis 143:A187 (1991)). In order to determine whetherspecific HIF sensitivity of the pulmonary vessels might be involved, thefollowing experiments were conducted to compare the contractileresponses of isolated pulmonary artery vessels (PA) from spontaneouslyhypertensive rats, SHR, and normotensive Sprague-Dawley rats. Theresults herein reported demonstrate that HIF is a potent vasoconstrictorof pulmonary arteries, and that this effect is significantly greater inspontaneously hypertensive rats than in normotensive Sprague-Dawleyrats. HIF appears to act by modulating the Na⁺, K⁺-ATPase-adrenergicneuroeffector interaction at the neuromuscular junction.

A. Preparation and Mounting of PA and Abdominal Aortic Rings

Adult male Sprague-Dawley (SD) and Spontaneously Hypertensive rats(SHR)(10-12 weeks, 300-400 g) were anesthetized with pentobarbitalsodium (50 mg/kg IP) and injected intraperitoneally with heparin. Asegment of abdominal aorta was excised, cut into rings 2-3 mm in length,threaded with dental wire and mounted in a 5 ml water jacketed organchamber filled with modified Krebs solution (MKS) and gassed with 93%O₂, 7% CO₂ at 37° C. (Malis, C. D., Leaf, A., Varadarajan, G. S.,Newell, J. B., Weber, P. C., Force, T. G., Bonventre, J. V., Circulation84:1393-1401 (1991)). Thoracotomy was then performed and rings from thelarge extrapulmonary right and left branches of the main pulmonaryartery (PA) were dissected free and mounted as for the aorta in aparallel tissue chamber. Oxygen and CO₂ tensions and pH were checkedsystematically by using a pH/blood gas analyzer. The functional statusof the endothelium in aortic and pulmonary artery rings was determinedby demonstrating acetylcholine-induced relaxation.

B. Experimental Protocol

HIF was prepared as described above in Example I. One unit of HIF in thevessel studies is defined as that amount of inhibitor that inhibitsouabain-sensitive K⁺ transport by 50%, as determined by ⁸⁶Rb⁺ uptakeinto human erythrocytes (Carilli, C. T., Berne, M., Cantley, L. C.,Haupert, G. T., Jr., J Biol Chem 260:1027-1031 (1985)). Because themolecular weights, and binding affinity of HIF and ouabain for purifiedNa⁺, K⁺-ATPase are similar (Haupert, G. T., Carilli, C., Cantley, L. C.,Am J Physiol 247:F919-F924 (1984)), it can be estimated from theerythrocyte assay that 1 unit HIF is approximately 0.75 pmolouabain-equivalent bioactivity. This estimate agrees very closely withearlier calculations that 1 unit/50 μl=15 nM HIF (Haupert, G. T.,Carilli, C., Cantley, L. C., Am J Physiol 247:F919-F924 (1984)).

Baseline resting tension of vascular rings was set at 1.5 g based onmaximal KCl-induced contractile response. Isometric force measurementswere obtained with a Grassforce displacement transducer and continuouslyrecorded. Pulmonary artery and aortic rings were studied simultaneously.Ring viability and contractile response was calibrated using KCl (5-25mM). Rings were then washed free of KCl, equilibrated to stablebaseline, and superfused with MKS containing HIF or ouabain. Wet weightof rings was obtained at the end of each experiment. The amplitude ofcontractions was measured as milligrams of tension above restingtension, percent change above resting tension, or change in tension inmilligrams per milligram wet weight tissue. Data are expressed asmeans±SEN. Analysis of variance followed by multiple comparison by theFisher test for multiple comparisons were used to determine differencesbetween groups. Paired Student's t tests were used where appropriate.Significance was assumed at p<0.05.

C. Effects of HIF on Resting Tension

HIF evoked a significantly greater contractile response in PA ofhypertensive rats compared to normotensive rats. When normalized to themean contractile response to a standard KCl concentration (15 mM),enhanced response in PA of SHR compared with SD remained highlysignificant (132±20% v. 35±6% of the KCl-stimulated tension, p<0.001).

The effect of HIF on PA was reversible and caused a cumulativeconcentration-dependent increase in tension in PA rings of hypertensiveand normotensive rats, whereas approximately equimolar concentrations ofouabain did not. Ouabain in even much higher concentrations (4×10⁻⁷M and10⁻⁴ M) did not elicit contractions in PA rings consistent with theknown resistance of rat Na⁺, K⁺-ATPase to ouabain. HIF-inducedcontractions were not altered in de-enothelialized aortic rings. HIF didnot increase the sensitivity of PA rings to exogenous norepinephrine ascontractile responses to different concentrations of norepinephrineranging from 10⁻⁹M to 10⁻⁶M were identical before and after addition ofHIF.

The effects of 20U HIF (approximately 4 nM) on resting tension ofpulmonary artery and aortic rings in SHR and SD are summarized in Table5.

Whereas HIF constricted PA rings of hypertensive rats to a significantlygreater extent than PA rings of normotensive rats, this difference wasnot observed in abdominal aortic rings (Table 5). In addition, forHIF-induced contractions, no significant difference was found betweenaortic and PA rings in normotensive animals, but in hypertensive ratsthe effect of HIF was significantly greater in PA compared to aorticrings (Table 5). This difference was not accounted for by a differencein vessel mass as change in tension normalized per milligram wet tissueweight was also significantly greater in PA rings compared with aorticrings (221±46 mg/mg wet wt vs. 108±19 mg/mg wet wt, respectively,p<0.05).

TABLE 5 HIF-induced change in tension (increase over resting tension) invarious isolated vascular rings. Vessel [HIF], nM ΔTension (mg) n p SHRPA 4 308 ± 56 8 <0.02 S-D PA 4 137 ± 26 8 SHR Ao 4 145 ± 29 6 = 0.22 S-DAo 4  93 ± 21 4 SHR PA 4 308 ± 56 8 <0.05 SHR Ao 4 145 ± 29 6 S-D Pk 4137 ± 26 8 = 0.3 S-D Ao 4  93 ± 21 4

SHR refers to spontaneously hypertensive rats; S-D efers toSprague-Dawley rats; Ao refers to aortic rings; nd PA refers topulmonary artery rings; n is the number of vascular rings studied.Tension values are means±SEM.

Pharmacologic Interventions

HIF-induced vasoconstriction in aortic and PA rings was completelyabolished by 10⁻⁶M phentolamine. FIGS. 5A-5B show the effects of thealpha-blocker phentolamine (PhA, 10⁻⁶M) (top), and nominal zero calcium(bottom) on HIF-induced constrictions of a representative vessel. At2×10⁻⁷M, the Ca²⁺ channel blocker, verapamil, reduced the HIF-inducedincrease over resting tension by 35% (range 31% to 38%). However,5×10⁻⁸M verapamil, which exclusively blocks the Ca²⁺ channels in PAwithout affecting alpha receptors, did not reduce the HIF-inducedcontraction.

HIF-induced increase in resting tension was dependent on extracellularCa²⁺. When Ca²⁺ was omitted from the MKS in the tissue bath, almost norise (<5%) over resting tension was observed with HIF (FIGS. 5A-5B).When Ca²⁺ was added back to the bath, tension promptly rose onretreatment with HIF.

Complete abolition of HIF-induced contractions in the presence of thealpha-receptor blocker phentolamine (1 μM) (FIGS. 5A-5B), suggests thatthe contraction of the smooth muscles was due mainly to the action ofnorepinephrine. The exocytotic release of norepinephrine into thejunctional cleft is normally triggered by Ca²⁺ entry into the nerveterminal following the propagation of the action potential along theneuronal cell membrane. Norepinephrine release is therefore inhibited ina zero calcium milieu, a condition which also eliminated HIF-inducedvasoconstriction (FIGS. 5A-5B). Readdition of calcium to bathing mediumcontaining HIF restored the vasoconstrictive response associated withHIF. HIF cannot be a catecholamine itself, since catecholamines produceinitial vasocontrictions in zero Ca²⁺ medium through direct stimulationof the alpha receptor and resulting release of Ca²⁺ from intracellularstores. Furthermore, as described in Example VII, the structure of HIFis now known to be that of a glycosylated steroid lactone.

On the other hand, voltage dependent calcium channel blockage withverapamil (2×10⁻⁷M) only caused a small decrease in HIF-induced tensiondevelopment, and had no effect at all at lower doses (5×10⁻⁸M). Thelatter concentration was shown in rabbit pulmonary artery to blocktransplasmalemmal Ca²⁺ entry through voltage dependent calcium channels(Haeusler, G., J Pharmacol Exp Ther 180: 672-682 (1972)), but is a dosetoo low to produce partial alpha blockade that can occur with the largerverapamil dose we first used. These results indicate that depolarizationof the vascular smooth muscle cell membrane with subsequent opening ofcalcium channels is not the likely mechanism by which HIF-induced sodiumpump inhibition evokes the mechanical response in PA.

The results herein reported can be explained by considering that therelease and reuptake of norepinephrine into sympathetic nerve terminalsis coupled to neuronal Na⁺ pump activity (Vanhoutte, P. M., Lorenz, R.R., J Cardiovasc Pharmacol 6:S88-S94 (1984)). Na⁺, K⁺-ATPase inhibitorsblock the neuronal uptake mechanism, and prevent post-junctional tissueaccumulation of norepinephrine as shown by increased accumulation oftritiated norepinephrine in the junctional cleft following cardiacglycoside treatment (Flaim, S. F., DiPetti, D. J. Am J Physiol236:613-619 (1979)). HIF-induced contractions may therefore result fromincreased concentrations of active neurotransmitter in the junctionalcleft, with enhanced activation of postjunctional smooth muscle cellalpha-receptor sites. This concept fits with the long recognized role ofthe sympathetic nervous system in the genesis and/or maintenance of thehypertensive state in rat models of spontaneous hypertension. Indeed,many parameters of sympathetic function in blood vessels of hypertensiveanimals are changed, and chemical denervation with 6-hydroxydopamine canprevent or attenuate the development of hypertension (Hallback, M.,Weiss, L. Med Clin North Amer 61:593-609 (1977)). In the SHR the Na⁺,K⁺-ATPase-coupled neuronal norepinephrine uptake process is activated,so that its efficacy in controlling the junctional concentration ofnorepinephrine is already enhanced (Vanhoutte, P. M., Verbeuren, T. J.,Webb, R. C., Physiol Rev 61:151-247 (1981)). Interference with thislocal modulatory mechanism by an endogenous inhibitor might therefore beexpected to have a more prominent pathophysiologic impact inspontaneously hypertensive rats than in the normotensive controls.

VII. Structural Analysis of HIF

Summary of Results:

Endogenous inhibitors of Na⁺, K⁺-ATPase have been isolated frommammalian sources and partially characterized by several laboratories.Recently, Mathews et al. (Hypertension 17:930-935 (1991)) reported thatthe factor they described was indistinguishable from ouabain based onHPLC coelution and mass spectrometric analyses. The mass spectroscopicmethods employed, however, could not permit assignment of precisestructure. Nonetheless, physiologic testing of the purified plasmainhibitor again gave results indistinguishable from ouabain (Bova, S.,Blaustein, M. P., Ludens, J. H., Harris, D. W., DuCharme, D. W. andHamlyn, J. M. Hypertension 17:930-935 (1991). In order to determinewhether the comound described herein, bovine hypothalamic factor (HIF),is distinguishable from ouabain, sophisticated structural analyses wereperformed. To provide enough purified HIF to perform these analyses, anaffinity purification procedure was developed that permitted a total of2,200 units of HIF to be purified in several batches for structuralanalysis.

Using affinity chromatography and reversed phase HPLC as the penultimateand final purification steps, HIF was purified to homogeneity.Coinjection of acyl derivatives of ouabain and pure HIF showed that thetwo molecules had different retention times on HPLC, indicating adifference in structure. Differences in circular dichroism spectrometryof the penta- and hexa napthoyl derivatives of HIF and ouabain confirmedthat there was difference in structure between these two compounds.Using the techniques of tandem mass spectrometry, circular dichroismspectrometry of acyl derivatives of HIF, gas chromatography-massspectrometry and liquid chromatography-mass spectrometry of the cleavedsugar and steroid moieties, exact molecular mass and specific structuralassignment became possible using submicrogram quantities of HIF. Wereport here that HIF is, like ouabain, an alpha L-rhamnoside, butdiffers from ouabain in its sugar-cardenolide arrangement, a changepresumed to account for the observed differences in biological activity.

Affinity-purified HIF was found to be indistinguishable from ouabain inmolecular mass. However the information generated ion spray tandem massspectrometry did not exclude the possibility that HIF is an isomer ofouabain. To address this uncertainty, ultramicrospectroscopic probes ofaffinity purified HIF were designed to unambiguously assign as much ofthe molecular structure possible given a limit of approximately onemicrogram of purified HIF as starting material. Two separate strategieswere developed to analyze HIF: one to define the nature of the presumedsugar moiety; and the second to assign the stereo- and regio-chemistryof the intact glycoside and aglycone. In both cases, the experimentalconditions were first established for authentic ouabain and then appliedto both ouabain and HIF in a side by side comparison.

In the case of identifying the sugar subunit, analysis of HIF alongsideouabain on a 10 pmole per experiment scale has demonstrated the presenceof rhamnose in both HIF and Ouabain. The intact glycosides were thenanalyzed on a larger scale (500 pmole) by derivatization, HPLC andcircular dichroism (CD). The appearance of different derivatives for HIFand ouabain in this experiment provides the first chemical evidence thatHIF is a structural isomer related to, but distinct from, ouabain.Because both HIF and ouabain are rhamnosides, the structuraldifference(s) could be due to a difference in the sugar stereochemistry,the position of glycosylation and/or the structure or stereochemistry ofthe aglycone of HIF as compared to ouabain.

A. Purification of HIF

Initial Purification: Initial purification of the Na⁺ /K⁺-ATPaseinhibitor was accomplished from bovine hypothalamus usingaqueous/organic extractions, lipophilic gel chromatography, and cationand anion exchange chromatographies as previously described (Carilli, C.T., Berne, M., Cantley, L. C. and Haupert, G. T. Jr., J. Biol. Chem.260:1027-1031 (1985)); Anner, B. M., Rey, H. G., Moosmayer, M.,Meszoely, I., and Haupert, G. T. Jr., Am. J. Physiol. 258:F144-F153(1990), and Example I above). Extracts purified to this point were freeof proteins, lipids, vanadate and cations known to interfere in Na⁺/K⁺-ATPase bioassays. Dry residue after ion exchange was taken up in 10ml of deionized water and applied to a 30 ml column of CHP20P resin(Mitsubishi) developed in water and eluted with a linear methanolgradient 0-100% pumped at 2 ml/min over 120 min. Na⁺ /K⁺-ATPaseinhibitory activity was monitored using ouabain-sensitive inhibition of⁸⁶Rb⁺ uptake into human erythrocytes, inhibition of purified Na⁺/K⁺-ATPase, and inhibition of ³H-ouabain binding to a microsomalpreparation of Na⁺ /K⁺-ATPase as previously described (Haupert, G. T.,Jr. and Sancho, J. N., Proc. Natl. Acad. Sci. USA 76:4658-4660 (1979);Haupert, G. T., Jr., Carrilli, C. T., and Cantley, L. C., Am. J.Physiol. 247:F919-F924 (1984); Carilli, C. T., Berne, M., Cantley, L. C.and Haupert, G. T., Jr., J. Biol. Chem. 26: 1027-1031 (1985)). Eluatesof the CHP20P column contained two inhibitors of Na⁺ /K⁺-ATPaseactivity, a non-specific, polar substance which eluted at 12 min, andHIF which eluted in a peak at 84 min. Residue in the HIF fraction wasgenerally unweighable by microbalance, and yield of HIF after this stepranged from 150-750 pmol ouabain equivalent bioactivity/kg of startingtissue (1 unit of ouabain-equivalent bioactivity ˜0.75 pmol HIF(Haupert, G. T., Jr., Carrilli, C. T., and Cantley, L. C., Am. J.Physiol. 247:F919-F924 (1984); Anner, B. M., Rey, H. G., Moosmayer, M.,Meszoely, I., and Haupert, G. T., Jr., Am. J. Physiol. 258:F144-F153(1990)), or 90-440 ng HIF/kg (see below).

Affinity Chromatoaraphy Purification of Partially Purified HIF: The HIFwas further purified by an affinity chromatography step which employedcoupling of purified canine renal Na⁺ /K⁺-ATPase (Haupert, G. T., Jr.,Carrilli, C. T., and Cantley, L. C., Am. J. Physiol. 247:F919-F924(1984)) to glutaraldehyde activated magnetic iron oxide particles havingreactive primary amino groups (BioMag 4100, Advanced Magnetics,Cambridge, Mass.). Three ml of BioMag 4100 were activated with 5%glutaraldehyde following the manufacturers specifications. 4 mg ofpurified canine renal medullary Na⁺ /K⁺-ATPase were agitated in couplingbuffer overnight at 4° C. with the activated particles. Separation ofparticles from supernatants was done with a magnetic bar. Unreactedaldehyde groups were quenched with glycine. An aliquot of the magneticparticle-Na⁺ /K⁺-ATPase complex was combined with CHP20P purified HIF(100 pmol HIF/2mg ATPase) in the presence of a binding buffer consistingof 10 mM MgCl₂, 2 mM H₃PO₄, and 20 mM imidazole, pH 7.4 containing 250mM sucrose and 1 mM EDTA. The mixture was agitated at room temperaturefor 3 hours. Binding of HIF to the batch column was documented bymonitoring disappearance of HIF activity from the supernatant asmeasured with the coupled enzyme assay (Haupert, G. T., Jr., Carrilli,C. T., and Cantley, L. C., Am. J. Physiol. 247:F919-F924 (1984)).Binding efficiency was calculated by comparing supernatant HIF activityat T=0 min and T=180 min (T₀-T₁₈₀=HIF bound). At 3 h, binding buffer wasseparated from the column, and the particle-enzyme-HIF complexresuspended in an eluting buffer composed of 5 uM EDTA in 7.5 mMimidazole, pH 7.4. The eluting mixture was agitated overnight at 37° C.,the supernatant separated from the column complex, and concentrated todryness.

HPLC Chromatographic Purification of Affinity Purified HIF: Dried HIFwas reconstituted in 250 μl of distilled water and injected onto aWaters Resolve RP C18 (8 mm×10 cm), flow rate 1 ml/min. A step gradientelution was employed: 100% H₂O:0% CH₃CN for 3 min, followed by a lineargradient which reached 87% H₂O:13% CH3CN by 4 min and was continued atthis mixture for 40 min. HIF activity eluted in a single peak at 18-21min. Active fractions were pooled, assayed in the ⁸⁶Rb⁺ transport assayto calculate total units recovered, and rechromatographed. Arepresentative HPLC chromatogram of rechromatographed affinity purifiedHIF is shown in FIG. 6. The fraction representing active sample wasconcentrated to dryness and stored under Argon gas at −70° C. until usedin structure analysis experiments.

B. Ion Spray-Tandem Mass Spectrometry

Ion spray/tandem mass spectrometry of HIF indicated that it isindistinguishable from ouabain in mass. FIGS. 7A and 7B show repeatanalyses on 2 different preparations of HIF compared with ouabain. Onlyions having an m/z of 602 are selected in the first quadrupole and onlydaughter ions having an m/z of 439 are selected in the third quadrupole.The detection of comparable ion intensity for HIF and ouabain as shownin FIGS. 7A and 7B indicates that the unknown sample must have anidentical mass to ouabain as well as a daughter ion with the same massas the steroid subunit of ouabain. The first two peaks shown in FIG. 7Aindicate a strong signal intensity for ouabain (peak B) or ouabainspiked with acetonitrile from a side fraction from the HPLC purificationof HIF (peak A). This fraction contains no active HIF but its presencewith ouabain dampens the signal intensity when compared with ouabainalone. The positions marked C and D show the lack of signal given byinjections of acetonitrile alone. Position E indicates the lack ofsignal given by the inactive side fraction used to dampen the ouabainsignal in injection A. The active fraction of HIF gave a clear signal asshown by the repeat injections labelled at positions F and G. Anadditional inactive side fraction is shown at position H. FIG. 7B showsa repeat series of injections with a different preparation of HIF.Position A indicates ouabain. Position B is another inactive sidefraction of acetonitrile from the HIF purification. Position C is HIFwhich is followed by an inactive side fraction labelled D. Position Eindicates ouabain spiked with acetonitrile.

Methods:

Purified HIF or ouabain was ionized by injecting either 50 units of HIFor 5 ng of ouabain into a mobile phase of acetonitrile/H₂O containing 10mM ammonium acetate. This chemical ionization procedure produces an(M+NH₄)⁺ ion for ouabain having an m/z of 602. This ion is mass selectedin the first quadrupole of a triple stage tandem mass spectrometer. Thision then undergoes collision with argon in the second quadrupole,resulting in a loss of the sugar moiety (as a neutral species) as wellas a loss of ammonia. What remains is the steroid portion of ouabainwith a positive charge having an m/z of 439. This ion is then massselected in the third quadrupole and is the only ion detected. Dissolvedsamples were taken up in a 0.05 ml syringe and infused into the ionizingchamber at an infusion rate of 0.003 ml/min using a Harvard Apparatussyringe pump, model number 22. Spectra were acquired on a Sciex API IIItriple quadrupole mass spectrometer. The electrospray voltage was +4800.Zero grade air was used as the nebulization gas at a pressure of 40 psi.

C. Analysis of the Sugar Moiety of HIF:

The results of ion spray/tandem mass spectrometry analysis wasconsistent with HIF containing a sugar with a molecular mass identicalto rhamnose. Using authentic ouabain as a model, conditions formicroscale cleavage of ouabain to release rhamnose from the steroid weredeveloped using both the enzyme naringinase (an α-L-rhamnosidase) andacid hydrolysis (Methods). The enzymatic cleavage experiments could bescaled down to the 10 pmol level by monitoring the extent of hydrolysisthrough bioassay of reaction mixture aliquots, since for both HIF andouabain removal of the sugar moiety to produce the respective geninsreduced biological activity by 100-fold in the ⁸⁶Rb⁺ assay (data notshown). Direct sugar analysis on the residual amount of enzymaticallycleaved HIF was not possible due to a high background level of manysugars (including rhamnose) found in the commercial preparation ofnaringinase. Thus, another 10 pmol each of HIF and ouabain werehydrolyzed with HCl. Persilylation of these hydrolysates and analysis byGC/MS showed that both HIF and ouabain release rhamnose upon hydrolysis.The GC/MS retention times for perisylated sugars are shown in Table 6below. It should be noted that several isobaric sugar derivatives (suchas fucose, deoxyglucose and deoxygalactose) were easily resolved underthe same GC/MS conditions.

The enzymatic hydrolysis experiments demonstrate that HIF containsrhamnose, but its identification as the D or L isomer could not beanswered unambiguously, although the stereospecificity of mostglycosidases supported the assignment of HIF as an α-L-rhamnoside.Definitive assignment of the isomeric form was therefore undertakenusing chiral GC/MS analysis of the HIF-derived rhamnose.Tetratrifluoroacetates of sugars were prepared and analyzed for D and Lrhamnose and acid hydrolysates of HIF (Methods). Table 7, below, showsretention times for the derivatized sugars. HIF hydrolysate and HIFhydrolysate spiked with L-rhamnose showed identical retention times andsingle peaks, while HIF hydrolysate spiked with D-rhamnose showed twopeaks representing the L-rhamnose of HIF and the added D isomer. HIF isthus concluded to be an a-L-rhamnoside.

TABLE 6 GC/MS Retention Times for Persilylated Sugars Retention Time forPersilylated Product(s) Undervatized Sugar (min) 7:01 L-rhamnose 7:527:34 L-fucose 8:04 7:45 2-deoxy-D-galactose 8:02 8:25 8:082-deoxy-D-glucose 9:04 8:27 quinovose 9:13 2,5-anhydro-D-mannitol 8:117:00 Ouabain acid-hydrolysate 7:53 6:59 HIF acid-hydrolysate 7:52

TABLE 7 Chiral GC/MS Analysis of Liberated Sugarts for HIF and OuabainSample Retention time (min:sec) Ouabain hydrolysate + 6:15 single peakauthentic L-rhamnose HIF hydrolysate 6:18 HIF hydrolysate + L-rhamnose6:17, single peak (first injection) HIF hydrolysate + L-rhamnose 6:19,single peak (second injection) HIF hydrolysate + 6:24 and 6:28D-rhamnose

Methods:

Enzymatic Hydrolyses

Naringinase (Sigma N-1385) was dissolved to make a 2 mg/ml solution in9:1 solution of 10 mM pyridinium acetate, pH 4.7/methanol. Subsamples(10 pmole per 50 μl of distilled water) of ouabain (Aldrich 14, 193-3)or affinity and HPLC-purified HIF were delivered into 100 μlmicrocentrifuge tubes. Naringinase solution (50 μl) was added to startthe reaction, then the tubes were sealed and incubated for 22 hours at37° C. Aliquots for bioassay measurements were removed (10 μl perdetermination, duplicate determinations for T=0 and T=22 hours) andevaporated under vacuum. The dry samples were reconstituted in 50 μl ofRb assay buffer for analysis.

Acid Hydrolyses:

Subsamples (10 pmole each in distilled water) of ouabain (Aldrich14,193-3) or affinity and HPLC-purified HIF were delivered into 100 μlmicroconical glass vials and dried in a Speed Vac concentrator. Thedried residues were redissolved in 50 μl of 2 N sequanal grade HCl,heated at 110° C. for 5 minutes in a dry block and then chilled withice. The acid was removed with a Speed Vac concentrator prior toderivatization.

Sugar Analysis:

Hydrolyzed samples or authentic sugar standards were prepared foranalysis by reaction with 10 μl N-trimethylsilyl imidazole (Pierce88623) at room temperature for 30 minutes. The resultant solutioncontaining persilylated sugars was analyzed directly by gaschromatograph/mass spectroscopy (GC/MS) using a 30 M DB-1 capillarycolumn (J&W Scientific 122-1032), and using a splitless injection at225° C. The column temperature was held at 120° C. for 1 minute, then a16° C./min linear ramp was applied from 120° C. to 180° C. followed by a5° C./min linear ramp from 180 to 250° C. The mass spectral measurementswere performed using ammonia chemical ionization and monitoring m/z 470& 380, (M+NH₄)⁺ and (M+NH₄)⁺—(CH3)₃SiOH respectively (these ions arecharacteristic for the persilylated sugars isobaric with rhamnose). Gaschromatograph retention times, for both alpha and beta anomers ofauthentic sugar standards, were obtained from the summed ion currenttraces.

Chirality Determination

Authentic L-rhamnose, D-rhamnose and hydrolysates were prepared foranalysis by reaction with a minimum volume ofN-methyl-bis-trifluoroacetamide (Pierce 49700) and pyridine (1:1, 10 μl)at 60° C. for 30 minutes to form sugar tetrakistrifluoroacetates.Reaction mixtures were cooled and analyzed directly by chiral GC/MSusing splitless injection (1 μl) onto a 25M Chirasil Val capillarycolumn (Alltech 13636). The GC injector temperature was 175° C. and thecolumn was held at 80° C. for 2 m, followed by a linear temperaturegradient from 80 to 150° C. at 5° C./min.

The mass spectral measurements were performed using methane chemicalionization and monitoring m/z 549 which corresponds to the (M+H)⁺ ionfor the tetrakistrifluoroacetate derivative of rhamnose. Chiralityassignments were confirmed by coinjection with reference standards.

D. HPLC Separation and CD Spectroscopy of Naphthoylated HIF and Ouabain

With HIF and ouabain having both identical molecular mass and the samesugar moiety, structural difference to explain differences in biologicalactivity were presumed to reside in the steroidal portion of themolecule. To further analyze the respective cardenolides the techniqueof circular dichroism spectroscopy of acylated derivatives was chosensince this method has been previously used to assign structure tonatural products at the microgram level. Preliminary studies onauthentic ouabain demonstrated that the choice of acylating reagents iscritical. Naphthoylation of 0.3 μg of ouabain formed the pentanaphthoateas the sole product, yielding enough product after preparative HPLC toprovide adequate signal for measurement of the CD spectrum.

Naphthoylations of ouabain and HIF were conducted side by side under thesame conditions. Analysis of the reaction products by HPLC showed thatouabain formed exclusively one product, as expected (the pentanaphthoylderivative, FIG. 8B), while HIF formed at least two products (FIG. 8A).Peak A derived from HIF showed a similar retention time as ouabainpentanaphthoate, while peak B derived from HIF is much more retained byRP HPLC than authentic ouabain hexanaphthoate (FIG. 8C). Afterpreparative HPLC, the product in peak A (FIG. 8A) from the HIFnaphthoylation reaction was shown to be distinct from ouabainpentanaphthoate by HPLC coelution (FIGS. 9A-9C). Although the massspectrum of component A was weak, a potential parent ion was observed aswell as fragment ions characteristic of ouabain pentanaphthoate (datanot shown). Based on retention time (lipophilicity), UV and CD spectra,and mass spectral daughter ion for the sugar naphthoate, component A ofHIF appears to be the pentanaphthoate, and component B, thehexanaphthoate of HIF. However the CD spectrum of component A (FIG. 10B)shows virtually no signal while a comparable amount of ouabain providesstrong exciton coupling with a positive Cotton effect (FIG. 11B). FIGS.10A and 11A show the corresponding ultraviolet absorbance spectra forHIF pentanaphthoate and ouabain pentanaphthoate, respectively. The morelipophilic product of the HIF naphthoylation reaction, peak B, was foundto consist of two components (B1 and B2) after further HPLC analysis(FIG. 12). Based on the mass spectral fragments and lipophilicity, it ismost likely that components B1 and B2 are alternative forms ofhexanaphthoyl HIF. In the case of ouabain, the hexanaphthoate isexpected to provide a weaker CD spectrum than the correspondingpentanapthoate (FIG. 11B). In contrast, the pentanaphthoate of HIF doesnot provide a strong CD spectrum (FIG. 10B) while the putative hexaderivatives of HIF allow exiton coupling to be observed. The CD spectraof B1 (FIG. 13B) and of B2 (FIG. 14B) were found to be of opposite sign,the former with a positive Cotton effect and the latter with a negativeCotton effect (FIGS. 13A and 14A show the ultraviolet absorbance spectraof peak B1 and B2, respectively, on the Vydac C18 profile of HIFhexanaphthoate). The HPLC and CD profiles of B1 and B2 are further proofthat HIF and ouabain are structurally different.

E. Characterization of the Aglycone of HIF by LC/MS

Side by side comparisons of naringinase-treated ouabain and HIF werecarried out using full scan mass spectra and single ion monitoringchromatography of the respective hydrolysates. Single ion monitoringchromatograms of HIF hydrolysates showed ions of R_(t) of 5.5 min. and6.1 min. Full scan mass spectra at these respective retention timesshowed all expected ions for the aglycone of HIF and the holomolecule.Thus, at R_(t)=5.5 min., parent ions were observed at m/z 439+ and 456+,representing (M+H)⁺ and (M+NH₄)⁺ ions of the HIP genin (FIG. 15A), andat R_(t)=6.1 min., parent ions were observed at 585+ and 602+,representing (M+H)⁺ and (M+NH₄)⁺ ions of uncleaved HIF (FIG. 15B).Retention times for the HIF-genin and holo-HIF coincided exactly withside by side analysis of the R_(t) for ouabagenin and authentic ouabain(data not shown). Since the full scan mass spectrum of HIF at a R_(t)coincident with reference ouabain was indistinguishable from ouabain,there is no evidence that HIF contains more than one sugar subunit.

Thus, in addition to containing L-rhamnose in an a glycosidic linkage,the aglycone of HIF is confirmed to have the same molecular mass asouabagenin with an identical retention time on HPLC. This resultindicates a remarkable similarity in the stereochemistry of the geninsof HIF and ouabain, even though the two holomolecules are shown to benon-identical by HPLC resolution and CD spectral analysis of acylderivatives. These results indicate that HIF differs from ouabain as asubtle isomer in the steroid backbone, or as an isomer in the glycosidiclinkage.

Methods:

Naphthoylation:

Each sample (ca. 500 pmole in a silylated, conical vial) was dissolvedin 250 μl of anhydrous acetonitrile, then 1.5 mg of naphthoylimidazoleand 0.4 μl of 1,8-diazabicyclo[5.4.0.]undec-7-ene (DBU) were added. Thereactions were stirred at room temperature for 3 hours and then quenchedby adding 1 ml of 20% aqueous acetonitrile. Reaction mixtures wereloaded onto C18 Sep Pak (Waters, Millipore Corp.) cartridges and washedsequentially with 2 ml of 20% acetonitrile, 8 ml of 40% acetonitrile, 5ml of 50% acetonitrile and finally with 5 ml of acetonitrile. The lastwashing was collected and subjected to HPLC analysis.

HPLC of Napthoylation Products:

Preparative chromatography was performed on a Phenomenex C18 column(4.6×250 mm, 10 μm) with isocratic elution using MeOH/H₂O 98:8 with aPerkin-Elmer Series 4 chromatograph delivering a flow of 1 ml/min. Thecolumn was monitored using a Shimazu RF-551 fluorescence detector withexcitation at 234 nm and emission monitoring at 374 nm. For coelutionexperiments of the pentanaphthoates, a Vydac C18 column (4.6×250 mm) wasemployed with isocratic acetonitrile/water 83:17 at a flow of 1 ml/min.The Vydac column with acetonitrile/water 82:18 at a flow of 1 ml/min wasused or preparative separation of component B1 from B2.

CD Spectroscopy:

CD spectra were obtained on a JASCO J-720 spectropolarimeter usingacetonitrile as solvent. A microcell attachment was employed asnecessary.

Liquid Chromatograph/Mass Spectroscopy of Hydrolyzed HIF and Ouabain

Samples were dissolved in 10 mM pyridinium acetate, pH 4.7-methanol(9:1), and injected onto a PLRP-S column (4.4×150 mm, PolymerLaboratories, Amherst, Mass.). The column was eluted with a 1 ml/minlinear gradient from 90:10 to 80:20 of 2 mM ammoniumacetate-acetonitrile using a Waters 600MS HPLC. The HPLC effluent wasconnected to the LC/MS interface of a Sciex API III mass spectrometer(Thornhill, Ontario, Canada) where the flow was split 20:1 reducing theflow into the ionspray source to 50 Ml/min. The mass spectrometer wasscanned from 350 to 1000 daltons in 2.8 s.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific embodiments of the invention described specifically herein.Such equivalents are intended to be encompassed in the scope of thefollowing claims.

What is claimed is:
 1. A composition which is purified to homogeneityand which is characterized by a mass spectrum depicted in FIG. 15B.
 2. Acomposition which is purified to homogeneity and which, afternaphthoylation, yields a CD spectrum of FIG. 10B, 13B or 14B.
 3. Acomposition purified to homogeneity which, when applied to a CHP20Pcolumn, elutes from the column at about 84 minutes using a linearmethanol gradient (0-100%) pumped at 2 ml/min. and which has a molecularweight of 584 g/mol.
 4. A pharmaceutical composition which is purifiedto homogeneity, comprising a therapeutic amount of a HypothalamicInhibitory Factor wherein said Factor: (a) has a molecular weight of 584g/mol; (b) is substituted by a rhamnoside; (c) elutes from a CHP20Pcolumn at about 84 minutes using a linear methanol gradient (0-100%)pumped at 2 m/min; (d) is a non-peptidic steroid; (e) specifically bindsNa, K ATPase reversibly and with high affinity; and (f) wherein saidFactor is not ouabain.
 5. A composition which is purified to homogeneityand which is formed by a method comprising the steps of: (a)methanol/water extraction; (b) petroleum ether and chloroformextraction; (c) lipophilic gel chromatography in methanol; (d)ion-exchange chromatography; (e) chromatography using CHP20P resin; (f)affinity purification using SDS-extracted Na, K-ATPase; and (g)reverse-phase C18-HPLC using a linear gradient of acetonitrile/water. 6.The pharmaceutical composition of claim 5 wherein said inhibitor isisolated from mammalian brain tissue.
 7. The pharmaceutical compositionof claim 6 wherein said inhibitor is isolated from bovine brain tissue.8. The pharmaceutical composition of claim 7 wherein said Na, K-ATPaseis kidney Na, K-ATPase.
 9. A glycosidic, non-peptidic HypothalamicInhibitory Factor which is purified to homogeneity, having a molecularweight of 584 g/mol, and a pharmaceutically acceptable carrier, whereinsaid factor is not ouabain.
 10. The pharmaceutical composition of claim9 wherein said Factor differs from ouabain in its sugar cardenolidearrangement.
 11. The pharmaceutical composition of claim 9 wherein saidFactor differs from ouabain in its sugar stereochemistry.
 12. Thepharmaceutical composition of claim 9 wherein said Factor differs fromouabain in its aglycone stereochemistry.
 13. The pharmaceuticalcomposition of claim 9 wherein said Factor differs from ouabain in itsglycosylation position.
 14. A composition consisting essentially ofHypothalamic Inhibitory Factor which is purified to homogeneity.